The present invention relates to a new method for the purification and quantification of viral particles. More particularly, the invention relates to a method of purifying and quantifying adenoviruses by ion-exchange chromatography. The invention also relates to a method of identifying various adenovirus serotypes.
Gene therapy is currently undergoing a remarkable development and various clinical studies in humans have been in progress since the first trials conducted in 1990. Among the methods commonly used for the transfer of genes, viral vectors have proved particularly promising, and adenoviruses occupy a key position among them.
The development of adenovirus vectors in gene therapy requires access to two types of technologies which are nowadays limiting for the production of viral stocks: the first is to have a method which is rapid, is highly sensitive and is very selective for the quantification of viral particles in samples obtained from the steps of constructing and amplifying the virus considered; this point is particularly important for the optimization of the method of producing viral stocks; the second is to have a method of purification which is reliable, reproducible, simple and can be easily extrapolated on the industrial scale for the purification of virus particles.
The production of clinical batches of adenoviruses remains a long procedure because of the number of transfection and amplification steps whose productivity is not optimized. Recombinant adenoviruses are usually produced by introducing viral DNA into an encapsidation line, followed by mechanical or chemical lysis of the cells after about two or three days of culture (the kinetics of the adenoviral cycle being from 24 to 36 hours). According to another variant, the culture is continued for a longer period (8 to 12 days), and the viruses are directly harvested in the supernatant after spontaneous release by a phenomenon of autolysis of the encapsidation cells (WO 98/00524).
Generally, between 2 and 7 amplification cycles are necessary to constitute the viral stocks. A major limitation to the optimization of the method of producing viral stocks lies in the methods of titrating the viral particles. Indeed, biological methods are methods which are relatively sensitive and accurate, but are particularly long to carry out (about 4 to 15 days depending on the assay used, i.e. transgene activity (tdu) or plaque production (pfu)). Faster analytical methods have been developed but they do not have a sufficient degree of precision and sensitivity when the titrations of viral particles have to be carried out, without prior purification, in lysates, crude cellular extracts or culture supernatants. That is why successive amplification cycles are carried out with multiplicities of infection (MOI) which are estimated roughly. The result is that the amplification steps are not very reproducible, or are even sometimes longer and/or more numerous than would be necessary with an optimized method. The rapid and precise determination of the titres of adenovirus solutions would make it possible to adjust the multiplicity of infection for each step so as to optimize the entire method of producing adenovirus stocks.
The method of quantifying viral particles should satisfy several conditions. In the first place, it should be sufficiently sensitive to allow the assay of viral particles in preparations which are dilute or which have a low titre (typically less than 1xc3x97109 viral particles per ml (vp/ml)) without resorting to a prior enrichment step. It should be possible to carry out the assay of the viral particles directly in lysates or crude preparations, without the need to carry out a purification step or a prior treatment. Furthermore, this method should allow a high selectivity in order to eliminate possible interference with the numerous compounds present in the crude cellular lysates or extracts and of which the proportions may vary depending on the culture conditions.
A quantitative analytical method based on anion-exchange chromatography has been described in the literature (Huygue et al., Human Gene Ther. 6: 1403-1416, 1995; P. W. Shabram et al., Human Gene Ther. 8: 453-465, 1997). This method, which has a detection limit of the order of 1xc3x97108 vp/ml, is applicable to the titration of purified viral particles. However, the sensitivity of this method decreases once the analysis is carried out on lysates or crude cellular extracts. The detection limit is estimated at 2 to 5xc3x97109 vp/ml in such samples and this method does not make it possible to quantify the adenoviral particles in very dilute and nonpurified preparations such as the lysates of cells infected during the virus transfection and amplification steps for which the adenoviral titre is typically of the order of 1xc3x97108 vp/ml to 1xc3x97109 vp/mi. Furthermore, neither does this method make it possible to quantify the adenoviral particles from preparations obtained in certain production media free of animal proteins. Indeed, such media contain, at the end of culture, compounds of the sugar, amino acid, vitamin or phenol red type, and the like, among which some may interfere with the adenoviral particles during the quantification of the virus and which lead to the titre of the preparation being very widely overestimated. Finally, the chromatographic method reported by Shabram et al. requires a pretreatment of the sample with a nuclease with a broad activity spectrum (BENZONASE) in order to remove the nucleic acids which interfere with the detection and measurement of the particles.
As regards the preparative methods of separating adenoviruses, chromatography has been used for many years for the purification of adenoviral particles [Haruna, I., Yaosi, H., Kono, R. and Watanabe, I. Virology (1961) 13. 264-267; Klemperer, H. G. and Pereira, H. G. Virology (1959) 9, 536-545; Philipson, L., Virology (1960) 10, 459-465]. Methods describing the large-scale purification of recombinant adenoviruses have been described more recently (international patent applications WO 96/27677, WO 97/08298, WO 98/00524, WO 98/22588).
Application WO 98/00524 describes in particular a method of purification using the strong anion-exchange resin Source 15Q which makes it possible to obtain, in a single chromatographic step, adenovirus preparations whose purity is at least equivalent to that obtained from preparations purified by caesium chloride gradient ultracentrifugation. This degree of purity is very high and reaches the standards required for clinical studies in humans (WHO Expert Committee on Biological Standardization, Forty-ninth Report. WHO Technical Report Series, WHO Geneva, in press).
However, when the viral titre of the preparations to be purified is low (for example in the case of an adenovirus having a low productivity, or when the purification has to be carried out using a stock obtained during an early amplification step), or alternatively when the virus production medium leads to the presence of compounds co-eluted with the adenovirus (as for example in the case of media free of calf serum), the limited performance of the chromatographic techniques previously described do not make it possible either to quantify or to purify the adenoviral particles in a single step from such a starting material.
The problem of being able to have a method of titrating viral particles from crude preparations which is rapid, sensitive and highly selective therefore exists. The problem of having a method of purification which is reliable, reproducible and makes it possible to obtain, from these same crude preparations, and preferably in a single step, viral preparations of pharmaceutical quality, also exists.
It has now been found, and this constitutes the subject of the present invention, that certain chromatography supports surprisingly exhibit quite exceptional properties for the separation of viral particles and in particular adenoviruses. These properties allow the titration and/or purification of viral particles from crude preparations, with no prior treatment, with a very high sensitivity and selectivity. The use of these supports provides, in addition and unexpectedly, a simple and rapid analytical method for separating and identifying, by chromatography, adenoviruses of different serotypes or adenoviruses modified at the level of the fibre or the hexon.
The subject of the present invention is a method of separating viral particles from a biological medium, characterized in that it comprises at least one chromatography step carried out on a support comprising a matrix and ion-exchange groups, the said groups being grafted onto the said matrix by means of a flexible arm.
The matrix may be chosen from agarose, dextran, acrylamide, silica and poly[styrene-divinylbenzene], alone or in the form of a mixture. Preferably, the matrix consists of agarose; still more preferably, it is approximately 6% cross-linked agarose.
The supports consisting of cross-linked agarose beads onto which are grafted functionalized, flexible ion-exchange arms have been developed for preparative and industrial chromatography of biomolecules. These supports have been more particularly designed for the step of capturing (that is to say the initial step of the method of purification) biomolecules from crude mixtures which have been simply clarified, that is to say freed of their solid constituents in suspension. Their performance was optimized in terms of a very high capacity for attachment of solutes to the support, of a very low counterpressure at a high linear liquid flow rate, of a low cost as well as of a very high chemical resistance to the cleaning agents used for regeneration.
Advantageously, the flexible arm is of a hydrophilic nature and it consists of a polymer of synthetic or natural origin. Among the polymers of synthetic origin, there may be mentioned the polymers consisting of monomers of polyvinyl alcohols, polyacrylamides, polymethacrylamides or polyvinyl ethers.
By way of polymer of natural origin, there may be mentioned in particular polymers of a polysaccharide nature chosen from starch, cellulose, dextran and agarose. Preferably, the degree of polymerization of the flexible arm is about 30 monomeric units, more preferably, the flexible arm is a dextran having a mean molecular weight of about 5000 Da.
Preferably, the flexible arm is functionalized by grafting a group capable of interacting with an anionic molecule. Most generally, the group consists of an amine which may be ternary or quaternary. Within the framework of the present invention, it is particularly advantageous to use a strong anion exchanger. Thus, a chromatography support as indicated above, functionalized by quaternary amines, is preferably used according to the invention.
By way of a support which is particularly preferred for carrying out the invention, there may be mentioned Q SEPHAROSE (Amersham Pharmacia Biotech). The use of this support is mentioned in one of the examples of application WO 98/39467. Purified adenoviruses are modified by treatment with polyethylene glycol (PEG). After reaction, the modified adenoviruses, the unmodified adenoviruses and the PEG are separated by passing over a Q SEPHAROSE XL column. This is therefore a simple separation between the starting materials and the final products of a chemical reaction. Persons skilled in the art could not assume that this column could be used successfully for the separation of adenoviruses from a complex biological medium containing various contaminating species (DNA from the host, RNAs, proteins, lipids, lipoproteins, endotoxins and the like), such as a lysate of encapsidation cells. Neither does it appear on reading this document that Q SEPHAROSE XL can be used for preparative purposes because it is known that the majority of supports lose their efficiency once large quantities of products are injected.
Other strong anion exchange supports having similar characteristics including matrix composition, particle size distribution, porosity, chemical nature of the flexible arm and grafting density may be used for the preparative or analytical separation of adenoviral particles. Advantageously, the matrix consists of 6% cross-linked agarose; it is grafted with flexible arms which consist of dextran and are functionalized with strong anion-exchange groups. The support has a particle size preferably of between about 40 and 200 xcexcm; the term xe2x80x9caboutxe2x80x9d relating to the particle size means that the value to be taken into consideration is within a deviation of between +/xe2x88x9220% relative to the value expressed. Preferably, this deviation is between +/xe2x88x9210% and more preferably it is between +/xe2x88x925% relative to the value expressed.
In the most particularly preferred manner, the particle size is between 45 and 165 xcexcm and is centred on 90 xcexcm.
Also advantageously, the matrix has a dispersion such that 95% of the particles have a diameter of between 0.1 and 10 times the mean diameter of the particles, and preferably between 0.3 and 3 times the mean diameter of the particles.
The Q SEPHAROSE XL used in the examples which follow illustrates nonexhaustively the performance of the supports which can be used within the framework of the invention.
The Q SEPHAROSE XL exhibits a bead size distribution ranging from 45 to 165 xcexcm, centred on 90 xcexcm. These size and bead distribution characteristics make the support a preparative-type chromatographic exchanger. Chromatographic theory and practice indicate that such a support has a very modest performance for the separation of compounds exhibiting similar chromatographic behaviour as regards the ion-exchange interaction. Likewise, such a support generates poorly resolved broad chromatographic peaks, in particular because of the large size and the very wide distribution of the beads constituting it. These expected chromatographic characteristics are verified for biomolecules in general such as proteins, which are eluted in the form of large, poorly separated peaks (see Data File Pharmacia Biotech No. 18-1123-82). On the other hand, in a completely unexpected manner, the adenovirus particles are eluted from this type of support in the form of a very symmetrical, extremely narrow peak. Compared with proteins, such as for example albumin, the efficiency of a column filled with Q SEPHAROSE XL, measured by the Height Equivalent to a Theoretical Plate (HETP) or the number of theoretical plates per unit of column length (N/m), is 50 to 100 times higher for adenovirus (N/m:35,000) than for proteins such as bovine serum albumin (N/m 600). See for example FIG. 1. Thus, when it is used under optimized chromatographic conditions, this type of gel, and in particular the Q SEPHAROSE Sepharose(copyright) XL gel, gives an adenovirus chromatographic peak whose narrowness is not equalled by the supports generally recommended for the separation of biomolecules. Among the supports recommended for the separation of biomolecules, there may be mentioned the supports whose basic matrix is of the poly[styrene-divinylbenzene] type (such as for example the resins Source 15Q and Source 30Q, or the resins of the Poros HQ, Poros DE2 or Poros D type). There may also be mentioned supports whose basic matrix is of the methacrylate-ethylene glycol copolymer type such as for example the resins Toyopearl DEAE, QAE and Super Q, or the resins of the Fractogel TMAE, TMAE HiCap, DMAE or DEAE type whose functional ion-exchange groups are situated on polyacrylamide-type linear polymeric chains grafted onto the matrix.
The efficiency of the supports used within the framework of the present invention for the separation of adenovirus particles leads to a very high sensitivity in the detection of the particles. Thus, when these supports are used in analytical chromatographic columns, the unexpected chromatographic behaviour of the viral particles makes it possible to quantify the adenovirus with a detection limit which is way below the detection limit of the methods previously described. This detection limit is at least ten times less than what makes it possible to achieve a detection limit of the order of 1xc3x97108 vp/ml in preparations of the crude cell lysate type and a detection limit of the order of 1xc3x97107 vp/ml for purified viral preparations.
This type of support also makes it possible to provide a very high selectivity towards the contaminants present in the samples to be analysed, such as for example proteins and nucleic acids. The proteins exist in the form of peaks which are very broad and which are eluted way before the viral peak. The nucleic acids are eluted from the column with a saline concentration which is substantially greater than the concentration necessary for the elution of the virus. This characteristic, which is very different from that obtained with the chromatographic method previously described (Huygue et al., Human Gene Ther. 6: 1403-1416, 1995) makes it possible to eliminate interference from this type of compounds with the viral peak. Finally, even when the preparations to be analysed contain species which are co-eluted with the viral particles, the highly specific form of the viral peak makes it possible to easily identify it and to carry out its quantification.
Thus, the supports used within the framework of the invention make it possible to identify and quantify very easily and with great accuracy the peak for the adenovirus when the latter is analysed using preparations containing a large quantity of proteins and nucleic acids. The quantitative analysis of the particles as well as the purification can also be carried out using preparations obtained with a wide variety of viral production media or with media free of constituents of animal origin, such as for example albumin, whether of bovine origin, human origin or yet another origin (FIG. 2). It is also important to observe that the method described in the present invention is applicable to the analysis of samples containing nucleic acids without prior treatment with a nuclease without affecting either the sensitivity or the selectivity of the method.
In this regard, another subject of the invention relates to the use of this type of support, and of Q SEPHAROSE XL in particular, for the preparative separation or the purification of viral particles, in particular adenoviruses, from biological media. Such a method of separation may optionally comprise a preliminary chromatography step on another support such as those used in the method which is the subject of application WO 98/00524, and in particular the resin Source 15Q. Such a preliminary step may prove advantageous in specific cases, for example if an excessively large quantity of contaminants is present in the biological medium.
Another subject of the invention relates to the use of this type of support, and of Q SEPHAROSE XL in particular, for the quantitative analysis or the titration of viral particles, in particular adenoviruses, from biological media.
The biological medium from which the purification or titration of the virus is carried out may be a supernatant of encapsidation cells producing the virus or a lysate of encapsidation cells, or a prepurified solution of the said virus. When the preparative separation or the purification of the viral particles is carried out using a supernatant of producing encapsidation cells or a lysate, it may be advantageous to carry out a preliminary ultrafiltration step; preferably this step is carried out by tangential ultrafiltration on a membrane having a cut-off of between 300 and 500 kDa.
The method of purification according to the invention makes it possible to obtain viral preparations of a high quality in terms of purity with high particle yields (of the order of 75 to 80%) in one step, using a stock which is dilute and/or very rich in contaminants, under production conditions which are fully compatible with industrial requirements and with regulations regarding the production of therapeutic molecules.
Another subject of the invention relates to a method of quantifying adenoviruses, characterized in that the viral particles are separated by chromatography on a Q SEPHAROSE XL type support and the quantity of adenovirus is determined by measuring the absorbance of the chromatographic fractions. The method of the invention allows an easier and more accurate monitoring of the kinetics of production, directly on homogeneous samples of supernatant, without pretreatment, which allows a better reproducibility and a better control of the methods of producing the stocks of viral particles.
The subject of the invention is also the use of a Q Sepharose(copyright) XL type chromatography support for the identification of various adenovirus serotypes. Indeed, and surprisingly, it has been observed that this type of support makes it possible to separate and identify, simply and rapidly, a wide variety of adenoviruses of various serotypes directly from a sample of viral production medium by determining the retention time and the ratio of the absorbance values at 260 nm and at 280 nm for the chromatographic peak.
As regards the use of chromatography supports within the framework of the present invention, the separation of viral particles for analytical or preparative purposes may be carried out by applying to the chromatography column a salt elution gradient or alternatively according to an isocratic mode, that is to say at constant saline concentration.
For the preparative methods, the chromatographic support may be used in a conventional type chromatography column or in a column suitable for high-performance chromatography systems, using for example the Q SEPHAROSE XL support, or alternatively in an expanded or so-called xe2x80x9cfluidizied bedxe2x80x9d system, using for example the STREAMLINE Q XL support. The size of the chromatographic column is determined as a function of the quantity of virus present in the starting material.
The viral preparation to be purified may be applied to the support in a buffer whose conductivity is such that the virus is not retained on the support whereas the nucleic acids are bound. Advantageously, the conductivity is adjusted to 45 mS/cm. This specific embodiment then makes it possible to separate, by simple filtration through the Q SEPHAROSE XL support, the virus from the nucleic acids obtained from the host cell contaminating the viral preparation.
The methods of assaying and purifying and characterizing the various serotypes described in the present invention can be applied to various types of viruses, and adenoviruses in particular, whether they are wild-type viruses or recombinant viruses carrying a transgene of interest.
In addition to the above features, the present invention also comprises other characteristics and advantages which will emerge from the following examples which are given by way of illustration and with no limitation being implied.